Non-porous diagnostic devices for the controlled movement of reagents

ABSTRACT

The assay devices, assay systems and device components of this invention comprise at least two opposing surfaces disposed a capillary distance apart, at least one of which is capable of immobilizing at least one target ligand or a conjugate in an amount related to the presence or amount of target ligand in the sample from a fluid sample in a zone for controlled fluid movement to, through or away the zone. The inventive device components may be incorporated into conventional assay devices with membranes or may be used in the inventive membrane-less devices herein described and claimed. These components include, flow control elements, measurement elements, time gates, elements for the elimination of pipetting steps, and generally, elements for the controlled flow, timing, delivery, incubation, separation, washing and other steps of the assay process.

This application is a continuation-in-part of application Ser. Nos.08/447,981 now U.S. Pat. No. 5,885,527, and 08/447,895 now U.S. Pat. No.6,019,944, each filed May 23, 1995, each of which are divisionals ofapplication Ser. No. 08/065,528, abandoned, filed May 19, 1993, whichwas a continuation-in-part of application Ser. No. 07/887,526 filed May21, 1992 now U.S. Pat. No. 5,458,852; each of which is incorporated byreference herein. Priority is claimed from each of these applications.

FIELD OF THE INVENTION

This invention relates to devices for conducting assays, includingqualitative, semi-quantitative and quantitative determinations of one ormore analytes in a single test format. Unlike assay devices of the priorart, the inventive assay devices described herein do not involve the useof bibulous materials, such as papers or membranes. The inventivedevices of the present invention rely on the use of defined surfaces,including grooved surfaces, and capillarity alone or in variouscombinations to move the test reagents. The inventive devices describedherein provide means for the controlled, timed movement of reagentswithin the device and do not require precise pipetting steps. Theconcepts and devices of the present invention are especially useful inthe performance of immunoassays of environmental and industrial fluids,such as water, and biological fluids and products, such as urine, blood,serum, plasma, spinal and amniotic fluids and the like.

BACKGROUND OF THE INVENTION

Over the years, numerous simplified test systems have been designed torapidly detect the presence of a target ligand of interest inbiological, environmental and industrial fluids. In one of theirsimplest forms, these assay systems and devices usually involve thecombination of a test reagent which is capable of reacting with thetarget ligand to give a visual response and an absorbent paper ormembrane through which the test reagents flow. Paper products, glassfibers and nylon are commonly used for the absorbent materials of thedevices. In certain cases, the portion of the absorbent membercontaining the test reagents is brought into contact, either physicallyor through capillarity, with the sample containing the target ligand.The contact may be accomplished in a variety of ways. Most commonly, anaqueous sample is allowed to traverse a porous or absorbent member, suchas porous polyethylene or polypropylene or membranes by capillaritythrough the portion of the porous or absorbent member containing thetest reagents. In other cases, the test reagents are pre-mixed outsidethe test device and then added to the absorbent member of the device toultimately generate a signal.

Commercially available diagnostic products employ a concentrating zonemethodology. In these products, such as ICON^(R) (HybritechIncorporated), TESTPACK™ (Abbott Laboratories) or ACCULEVEL^(R) (SyvaCorporation), the device contains an immunosorbing or capture zonewithin a porous member to which a member of a specific binding pair isimmobilized. The surface of the porous member also may be treated tocontain one or more elements of a signal development system. In thesedevices, there is a liquid absorbing zone which serves to draw liquidthrough the immunosorbing zone, to absorb liquid sample and reagents andto control the rate at which the liquid is drawn through theimmunosorbing zone. The liquid absorbing zone is either an additionalvolume of the porous member outside of the immunosorbing zone or anabsorbent material in capillary communication with the immunosorbingzone. Many commercially available devices and assay systems also involvea wash step in which the immunosorbing zone is washed free ofnon-specifically bound signal generator so that the presence or amountof target ligand in the sample can be determined by examining the porousmember for a signal at the appropriate zone.

The devices described herein do not use bibulous or porous materials,such as membranes and the like as substrates for the immobilization ofreagents or to control the flow of the reagents through the device. Adisadvantage of, for example, membranes in diagnostic devices is that onboth microscopic and macroscopic scales the production of membranes isnot easily reproducible. This can result in diagnostic devices whichhave differential properties of non-specific binding and flowcharacteristics. The time gates of this invention can, however, beembedded in membranes or used in devices with membranes. Membranes arevery susceptible to non-specific binding which can raise the sensitivitylimit of the assay. In the case of immunochromatographic assay formatssuch as those described in U.S. Pat. Nos. 4,879,215, 4,945,205 and4,960,691, the use of membranes as the diagnostic element requires aneven flow of reagents through the membrane. The problem of uneven flowof assay reagents in immunochromatographic assays has been addressed inU.S. Pat. Nos. 4,756,828, 4,757,004 and 4,883,688, incorporated hereinby reference. These patents teach that modifying the longitudinal edgeof the bibulous material controls the shape of the advancing front. Thedevices of the current invention circumvent these membrane associatedproblems by the use of defined surfaces, including grooved surfaces,capillarity, time gates, novel capillary means, including channels andnovel fluid flow control means alone or in various combinations, all ofwhich are constructed from non-absorbent materials. In a preferred modeof this invention, the capillary channel of the diagnostic element iscomposed of grooves which are perpendicular to the flow of the assayreagents. The manufacture of grooved surfaces can be accomplished byinjection molding and can be sufficiently reproducible to providecontrol of the flow of reagents through the device.

In addition to the limitations of the assay devices and systems of theprior art, including the limitations of using absorbent membranes ascarriers for sample and reagents, assay devices generally involvenumerous steps, including critical pipetting steps which must beperformed by relatively skilled users in laboratory settings.Accordingly, there is a need for one step assay devices and systems,which, in addition to controlling the flow of reagents in the device,control the timing of the flow of reagents at specific areas in thedevice. In addition, there is a need for assay devices which do notrequire critical pipetting steps but still perform semi-quantitative andquantitative determinations. The invention devices and methods of thisinvention satisfy these needs and others by introducing devices which donot require precise pipetting of sample, which do not use absorbentmembers, which include novel elements called time gates for thecontrolled movement of reagents in the device and which are capable ofproviding quantitative assays.

Definitions

In interpreting the claims and specification, the following terms shallhave the meanings set forth below.

Target ligand--The binding partner to one or more receptors.

Ligand--Binding partner to aligned receptor.

Ligand Analogue--A chemical derivative of the target ligand which may beattached either covalently or non-covalently to other species, forexample, to the signal development element. Ligand analogue and targetligand may be the same and both are capable of binding to the receptor.

Liquid Analogue Conjugate--A conjugate of a ligand analogue and a signaldevelopment element;

Signal Development Phase--The phase containing the materials involvingthe signal development element to develop signal, e.g., an enzymesubstrate solution.

Receptor--Chemical or biochemical species capable of reacting with orbinding to target ligand, typically an antibody, a binding fragment, acomplementary nucleotide sequence of a chelate, but which may be aligand if the assay is designed to detect a target ligand which is areceptor. Receptors may also include enzymes or chemical reagents thatspecifically react with the target ligand.

Ligand Receptor Conjugate--A conjugate of a ligand receptor and a signaldevelopment element.

Signal Development Element--The element which directly or indirectlycauses a visually or instrumentally detectable signal as a result of theassay process. Receptors and ligand analogues may be bound, eithercovalently or noncovalently to the signal development element to form aconjugate. The element of the ligand analogue conjugate or the receptorconjugate which, in conjunction with the signal development phase,develops the detectable signal, e.g., an enzyme.

Reaction Mixture--The mixture of sample suspected of containing targetligand and the reagents for determining the presence or amount of targetligand in the sample, for example, the ligand analogue conjugate or thereceptor conjugate. As used herein the Reaction Mixture may comprise aproteinaceous component which may be the target, a component of thesample or additive (e.g., serum albumin, gelatin, milk proteins).

Ligand Complement--A specialized ligand used in labelling ligandanalogue conjugates, receptors, ligand analogue constructs or signaldevelopment elements.

Ligand Complement Receptor--A receptor for ligand complement.

Ligand Analogue-Ligand Complement Conjugate--A conjugate composed of aligand analogue, a ligand complement and a signal development element.

Capture Efficiency--The binding efficiency of the component orcomponents in the reaction mixture, such as the ligand analogueconjugate or the receptor conjugate, to the capture zone of thediagnostic element.

Capture Zone--The area on the diagnostic element which binds at leastone component of the reaction mixture, such as the ligand analogueconjugate or the receptor conjugate.

Capillarity--The state of being capillary or the exhibition of capillaryaction. Capillarity can be affected by the solid surface or the liquidsurface or both.

Biosensor--Any electrochemical, optical, electro-optical oracoustic/mechanical device which is used to measure the presence oramount of target ligands. For example, electrochemical biosensorsutilize potentiometric and amperometric measurements, optical biosensorsutilize absorbance, fluorescence, luminescence and evanescent waves.Acoustic/mechanical biosensors utilize piezoelectric crystal resonance,surface acoustic waves, field-effect transistors, chemical field-effecttransistors and enzyme field-effect transistors. Biosensors can alsodetect changes in the physical properties of solutions in which receptorbinding events take place. For example, biosensors may detect changes inthe degree of agglutination of latex particles upon binding antigen orthey may detect changes in the viscosity of solutions in response toreceptor binding events.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partially schematic, top perspective view of a device inaccordance with the present invention.

FIG. 1a is a partially schematic, perspective exploded view of thedevice showing the detail in the area of the sample addition reservoir,the sample-reaction barrier, the reaction chamber, the time gate and thebeginning of the diagnostic element.

FIG. 1b is a partially schematic, perspective exploded view of thedevice showing the detail in the area of the optional reagent reservoir,the sample addition reservoir, the sample-reaction barrier, the reactionchamber, the time gate and the beginning of the diagnostic element.

FIG. 1c is a partially schematic, perspective exploded view of thedevice showing the detail in the area of the optional reagent reservoirin fluid contact with the sample addition reservoir and the reactionchamber

FIG. 1d is a partially schematic perspective cutaway view of the flowcontrol means.

FIG. 2 is a partially schematic, perspective view of a second device inaccordance with this present invention, which may be used to addpre-mixed reaction mixtures.

FIGS. 3a-3b are partial schematic top view of the diagnostic elementshowing some potential placements of capture zones.

FIG. 4 is a partially schematic, perspective view of a used reagentreservoir.

FIG. 5 is a partially schematic view of embodiments of these deviceswhich are columnar or have curved opposing surfaces.

FIGS. 6a-6f are top views of time gates.

FIGS. 7a-7f show typical dimensions for a preferred time gate.

FIGS. 8a-8f are top views of sequential time gates.

SUMMARY OF THE INVENTION

The assay devices, assay systems and device components of this inventioncomprise at least two opposing surfaces disposed a capillary distanceapart, at least one of which is capable of immobilizing at least onetarget ligand or a conjugate in an amount related to the presence oramount of target ligand in the sample from a fluid sample in a zone forcontrolled fluid movement to, through or away the zone. The inventivedevice components may be incorporated into conventional assay deviceswith membranes or may be used in the inventive membrane-less devicesherein described and claimed. These components include flow controlelements, measurement elements, time gates, elements for the eliminationof pipetting steps, and generally, elements for the controlled flow,timing, delivery, incubation, separation, washing and other steps of theassay process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to diagnostic testing devices fordetermining the presence or amount of at least one target ligand. FIG. 1shows a preferred embodiment of a device 10 according to the invention.Generally, the devices of the invention have thicknesses of about 2 mmto 15 mm, lengths of about 3 cm to 10 cm and widths of about 1 cm to 4cm. The dimensions may be adjusted depending on the particular purposeof the assay. One device of this invention, as depicted in FIG. 1,generally illustrates some features of the inventive devices andportions of devices herein disclosed and claimed. The device 10comprises various elements, a sample addition zone 1, a sample additionreservoir 2, a sample reaction barrier 3, a reaction chamber 4, a timegate 5, a diagnostic element 6, and a used reagent reservoir 7. Thedevices are comprised of capillary channels which are formed when a topmember 8 is place don the bottom member 9 a capillary distance apart andwhich move the reagents and sample throughout the device. The top andbottom members may be married, the various chambers sealed and thecapillaries formed by a number of techniques, including but not limitedto, gluing, welding by ultrasound, riveting and the like. The elementsof the device can be used in various combinations with the diagnosticelement 6 to achieve a variety of desired functions. As one skilled inthe art will recognize these elements may be combined to performone-step or multistep assays. The devices 10 may also be used in theformation of reaction mixtures for the assay process. The device 20 inFIG. 2 may be used to add pre-mixed reaction mixtures for the generationof signal which relates to the presence or amount of the target ligand.

An optional reagent chamber 17 may be incorporated into device 10 or 20as depicted in FIG. 1b and FIG. 1c. The devices 10 and 20 may also beused with an optional fluid control means 18 as shown in FIG. 1d.

Features include, but are not limited to: 1) diagnostic elements whichare not comprised of bibulous materials, such as membranes, 2) means tocontrol the volume of sample or reaction mixture, 3) time gates, 4)novel capillary means, termed fingers herein and 5) novel flow controlmeans, sometimes referred to as a "gap" herein and 6) used reagentreservoir which prevents backward flow of reagents. Those of skill inthe art will appreciate that these elements are separately novel andnonobvious, and may be incorporated into diagnostic devices in variouscombinations and may be used with other elements known to those skilledin the art to achieve novel and nonobvious diagnostic test devices andheretofore unrealized benefits.

Each of the elements of devices 10 and 20 will be described separately,then representative descriptions of the devices of this invention willfollow.

Sample Addition Zone

Referring to FIGS. 1 and 2, the sample addition zone 1 of the devices 10and 20 is the area where sample is introduced to the device. The sampleaddition zone 1 can be a port of various configuration, that is, round,oblong, square and the like or the zone can be a trough in the device.

Sample Addition Reservoir

Referring to FIGS. 1 and 2, the sample addition reservoir 2 is anelement of the device which receives the sample. Referring now to FIG.1, the volume of the sample addition reservoir 2 should be at least thevolume of the reaction chamber 4 or greater. The sample additionreservoir 2 can be a capillary space or it can be an open trough. Inaddition, a filter element can be placed in or on the sample additionreservoir 2 to filter particulates from the sample or to filter bloodcells from blood so that plasma can further travel through the device.In a preferred embodiment, the volume or capacity of the sample additionreservoir 2 is 1 to 5 times the volume of the reaction chamber 4. Ingeneral, one selects a volume or capacity of this reservoir 2 such thatif the excess sample is used to wash the diagnostic element 6 thenenough volume of sample is needed to thoroughly remove any unboundreagents from the diagnostic element 6 arising from the assay process.This reservoir 2 may also contain certain dried reagents which are usedin the assay process. For example, a surfactant can be dried in thisreservoir 2 which dissolves when sample is added. The surfactant in thesample would aid in the movement of the sample and reaction mixturethrough the device by lowering the surface tension of the liquid. Thesample addition reservoir 2 is generally in direct fluid contact withthe sample-reaction barrier 3 (FIG. 1) or the diagnostic element 6 (FIG.2).

Sample-Reaction Barrier

As depicted in FIG. 1, the sample-reaction barrier 3 separates thesample in the sample addition reservoir 2 from the reaction mixture inthe reaction chamber 4. The sample-reaction barrier is desired becauseit provides the device with the capability of forming a precise reactionmixture volume. A precise volume of the reaction mixture is generallynecessary for assays in which semi-quantitative or quantitative resultsare desired. Thus, a precise pipetting step of the sample to the deviceis not required because the sample reaction barrier forms a reactionchamber of precise volume into which the sample is capable of flowing.The sample reaction barrier 3 is desired because the reactions whichtake place in the reaction chamber 4 should preferably be separated formthe excess sample in the sample addition reservoir 2. The samplereaction barrier 3 comprises a narrow capillary, generally ranging fromabout 0.01 mm to 0.2 mm and the surfaces of the capillary can be smoothor have a single groove or a series of grooves which are parallel orperpendicular to the flow of sample. In a preferred embodiment of thesample reaction barrier 3, now referring to FIG. 1a, grooves 12,parallel to the flow of sample, are incorporated onto one surface of thedevice a capillary distance, for example, 0.02 mm to 0.1 mm, from theother surface. The volume of sample which fills the sample-reactionbarrier 3 (FIG. 1a) should be kept to a minimum, from about 0.01% to 10%of the reaction chamber 4 volume so that the reagents of the reactionchamber 4 do not significantly diffuse back into the sample in thesample addition reservoir 2. That is, the diffusion of the reactionmixture back into the excess sample should be kept to a minimum so thatthe chemical or biochemical reactions occurring in the reaction mixtureare not substantially influenced by the excess sample in the sampleaddition reservoir 2. Groove depths can range from about 0.01 mm to 0.5mm and preferably from about 0.05 mm to 0.2 mm. When more than onegroove is used for this element, the number of groove is this element istypically between 10 and 500 grooves per cm and preferably from about 20to 20 grooves per cm. Sample from the sample addition reservoir 2 flowsover the grooves 12 by capillary action and then into the reactionchamber 4. In a further preferred embodiment, grooves, hereafter termed"fingers" 16, are situated in the wall of the reaction chamber 4 influid contact with the grooves 12 or capillary space of thesample-reaction barrier 3. These fingers 16 are typically 0.5 mm to 2 mmwide, preferably 1 mm to 1.5 mm wide and typically 0.1 mm to 1.5 mm indepth, preferably about 0.2 to 1 mm in depth. The fingers 16 in the wallof the reaction chamber 4 aid in the capillary flow of the sample intothe reaction chamber 4. That is, the fingers allow fluid to move from acapillary where the capillarity is relatively high to a capillary wherethe capillarity is lower. Thus, the capillary at the sample-reactionbarrier is generally more narrow and has a greater capillarity than thecapillary or space of the reaction chamber. This difference incapillarity can cause the flow of sample or fluid in the device to stopin the sample-reaction barrier capillary. Presumably, the fingers breakthe surface tension of the fluid at the interface of the two capillariesor spaces and thereby cause the fluid to move into a capillary or spaceof lower capillarity. One can appreciate that the utility of fingers canbe extended to any part of the device where fluid must flow from highcapillarity to low capillarity. In practice, this is usually when thedirection of fluid flow is from a narrow capillary (higher capillarity)to a wider capillary (lower capillarity). The top surface of the samplereaction barrier may also be used to immobilize reagents used in theassay process such that the sample flows over the sample reactionbarrier, dissolves the reagents and moves into the reaction chamber. Themovement of the sample and reagents into the reaction chamber may act asa mixing means.

Reaction Chamber

Referring to FIG. 1, the sample moves into the reaction chamber 4 fromthe sample-reaction barrier 3. The reagents of the device 10 arepreferably placed in the reaction chamber 4, for example, as dried orlyophilized powders, such that when the sample enters the reactionchamber 4 the reagents quickly reconstitute. The volume of the reactionchamber 4 is the volume of sample which defines the reaction mixture.The reaction chamber may be sealed on 2 sides, for example, byultrasonic welding of the top and bottom members. Thus, delivery of thesample to the device 10 at the sample addition zone 1 does not require aprecise pipetting step to define the volume of the reaction mixture.Mixing features which mix the reaction mixture can also be incorporatedin conjunction with the reaction chamber element 4, such as thosedescribed in U.S. patent application Ser. No. 711,621 filed Jun. 5,1991, hereby incorporated by reference. The sample fills the reactionchamber 4 because of capillary forces and also, potentially, because ofthe hydrostatic pressure exerted by the sample in the sample additionreservoir 2. The floor of the reaction chamber 4 may be smooth orcomprised of a grooved surface to aid in the movement of the sample intothe reaction chamber 4. The volume of the reaction chamber 4, andthereby the reaction mixture, may be any volume which accommodates thereagents and which provides the desired sensitivity of the assay. Theshape of the reaction chamber 4 should be such that the movement of thereaction mixture from the reaction chamber 4 is not turbulent and eddiesare not formed as a result of the movement out of the reaction chamber4. A preferred shape of the reaction chamber 4 is shown in FIG. 1. Thedepth of the reaction chamber 4 should be commensurate with the width ofthe chamber to accommodate the desired reaction mixture volume. Thedepth of the reaction chamber can range from about 0.05 mm to 10 mm andpreferably from 0.1 mm to 0.6 mm. To accommodate a particular volume ofthe reaction chamber, the length and width of the reaction chambershould be adjusted and the depth maintained as narrow as is practical.The reaction chamber 4 is in direct fluid contact with thesample-reaction barrier 3 and the diagnostic element 6 of time gate 5.In addition, the reaction chamber 4 may also be in direct fluid contactwith an optional reagent reservoir 17 as shown in FIGS. 1b and 1c.

A preferred embodiment of the reaction chamber utilizes a ramp whichextends from the bottom of the reaction chamber to the surface of thediagnostic element. The ramp minimizes or prevents mixing and eddieformation of the reaction mixture with the sample at the interface ofthe reaction chamber and the diagnostic element as the fluid movesthrough the device. Thus, the ramp allows a smooth transition of thefluid out of the reaction chamber and onto the diagnostic element. Thelength of the ramp should be optimized for each depth of the reactionchamber, but generally, the ramp is at an angle of between 25 and 45degrees relative to the floor of the reaction chamber.

Time Gate

Referring to FIG. 1a, the time gate 5 holds the reaction mixture in thereaction chamber 4 for a given period of time. The concept of the timegate is that a predominantly aqueous solution cannot pass through asufficiently hydrophobic zone until the hydrophobic zone is madesufficiently hydrophilic. Furthermore, the hydrophobic zone ishydrophilic through the binding of a component in the aqueous solutionto the hydrophobic zone. The sufficiently hydrophobic zone is generallyin a capillary space. The driving force for fluid movement over orthrough the time gate may be either the capillarity of the space orhydrostatic pressure exerted by the sample or a combination of both ofthese forces. The amount of time which is required to hold the reactionmixture in the reaction chamber 4 is relative to the assay process suchthat the reactions which occur in the reaction chamber 4 as a result ofthe assay process will reflect the presence or amount of target ligandin the sample. Thus, the time gate 5 delays the flow of the reactionmixture onto the diagnostic element 6. The time gate 5 delays the flowof the reaction mixture by the principle that a hydrophilic liquid, suchas an aqueous solution or one which has a dielectric constant of atleast 40, cannot move past a sufficiently hydrophobic barrier in acapillary channel. In designing and building a time gate, one can beginwith a hydrophobic surface, such as are found on native plastics andelastomers (polyethylene, polypropylene, polystyrene, polyacrylates,silicon elastomer sand the like) or silicon chip surfaces or metalsurfaces, either smooth, grooved or textured and a capillary is formedby an opposing surface which can be hydrophobic or hydrophilic in natureand smooth, grooved or textured. The hydrophobic surface(s) in thecapillary have a microscopic surface area onto which can bind componentswhich are generally soluble in a predominantly aqueous solution. Thehydrophilic character and the concentration of the component(s) in thereaction mixture and the overall surface area of the time gate affectsthe mechanics of the time gate. The amount of time for which the timegate 5 holds the reaction mixture is melted to the rate of binding of acomponent(s) from the reaction mixture to the hydrophobic barrier. Thebinding of the component(s) from the reaction mixture changes thehydrophobic barrier to a zone which is sufficiently hydrophilic overwhich or through which the reaction mixture can flow. Creating thesufficiently hydrophilic surface then allows the fluid to flow as if thetime gate had not been in the device. Thus, fluid flow through theremainder of the device is not affected once the time gate has been madehydrophilic. Other devices described which incorporate fluid delaymeans, for example, in U.S. Pat. Nos. 4,426,451 and 4,963,498, herebyincorporated by reference only, require an external manipulation of thedevice to start fluid flow or utilize capillary constrictions to slowfluid flow. In this latter case, the capillary constriction used todelay fluid flow will affect the fluid flow through the remainder of thedevice.

In a preferred embodiment, for example, the time gate 5 can be composedof latex particles 15 (FIG. 1a, not drawn to scale), such as polystyrenelatexes with diameters of between about 0.01 μm and 10 μm or hydrophobicpolymers, such as polypropylene, polyethylene, polyesters and the like,which are introduced onto the device in the appropriate zone where thereaction mixture must travel. In another preferred embodiment, the timegate can be created by applicatiin of a hydrophobic chemical, such as anink or a long chain fatty acid, or a hydrophobic decal to the desiredzone. The hydrophobic chemical or decal is generally not soluble or ispoorly soluble in the reaction mixture. In yet another preferredembodiment, the time gate can also be formed by changing a hydrophilicsurface to a hydrophobic surface. For example, hydrophobic surfaces madehydrophilic by plasma treatment can be converted back to a hydrophobicsurface by the application of solvents, ultraviolet light or heat andthe like. These treatments can act to change the molecular structure ofthe hydrophilic, plasma modified surface back to a hydrophobic form.

The component(s) in the reaction mixture which bind to the hydrophobiczone may be various proteins, polypeptides, polymers or detergents. Apreferred protein is bovine serum albumin. The time delay provided bythe time gate 5 depends on the concentration of the component(s) in thereaction mixture, for example, bovine serum albumin, which binds to thehydrophobic zone, for example, the surface area provided by the latexparticles 15. Another preferred embodiment of the time gate 5 utilizespolyelectrolytes which are hydrophobic and which become hydrophilic byexposure to the buffering capacity of the reaction mixture. The timegate 5 would be comprised of, for example, polyacrylic acid, which inits protonated form it is hydrophobic. The reaction mixture, if bufferedabove the pK_(a) of the polyacrylic acid, would deprotonate the acidgroups and form the hydrophilic salt of the polymer. In this case, thetime delay is related to the mass of polyelectrolyte and the pH and thebuffering capacity of the reaction mixture.

The geometry or shape of the time gate can influence the area of thetime gate that the fluid will pass over or through. That is, the timegate can be designed to direct the flow of liquid through a specificarea of the time gate. By directing the fluid to flow through a definedarea of the time gate the reproducibility of the time delay is improved.FIG. 6 shows representative geometries of time gates. For example, asshown in FIG. 6, time gates a-d, the time gates have V-shapesincorporated into their design, and more specifically, the length of thetime gate (defined as the distance the fluid must cross over or throughin order to pass the time gate) is less at the tip of the V than in thebody of the time gate. Thus, in a preferred mode, the fluid will crossover or pass through the time gate where the length is shortest therebydirecting fluid flow through the time gate in a consistent manner. Ingeneral, the directionality of fluid flow over or through the time gatesis represented by opposing arrows in FIG. 6. In a preferred embodiment,the orientation of the tie gates b, c and d of FIG. 6 are such that thefluid touches the flat portion of the time gate first rather than the Vshape. In other words, the preferred direction of flow for the timegates b, c and d of FIG. 6 is represented by the up arrow. In caseswhere the time gate is simply a line, for example as seen in FIG. 6,time gate e and f, the path of fluid flow over or through the time gatecan occur at any point on the time gate. Thus, the time gates which havegeometries directing the fluid flow over or through a consistent area ofthe time gate are preferred. For example, time gates with lengthsranging from about 1.3 mm to 0.13 mm achieve delay times ofapproximately 0.3 min to 5.5 min, respectively, when the distancebetween surfaces is about 0.018 mm. When the time gate is V-shaped thelength of the time gate at the tip of the V has dimensions smaller thanthe length of the time gate at the remaining portion of the V; that is,the arms of the V should have a length roughly 2 to 5 times the lengthof the V tip, as for example, FIG. 7, time gate a, illustrates. FIG. 7,time gate b, shows that only a small area of the time gate is crossedover or through at the tip of the V as compared with the remainder ofthe time gate. The time gate should span the width of the capillary orspace so that the entire fluid front comes in contact with the timegate. If the time gate was not as wide as, for example, the diagnosticelement, then the fluid front would go around the time gate. Thus, thetime gate should "seal" the fluid in the space during the delay period.

Referring to FIG. 1, one skilled in the art can recognize that eachdevice 10 could incorporate one or more time gates to achieve thedesired function of the device. FIG. 8 shows some examples of thesequential placement of several time gates of FIG. 6. For example, asdiscussed in the next section, Optional Reagent Chambers, if asequential addition immunoassay was to be performed by the device than 2time gates would allow 2 sequential incubation steps to be performed bythe device prior to the movement of the reaction mixture to thediagnostic element. In another example, if an incubation of the reactionmixture on the capture zone or zones of the diagnostic element(s) 6 wasrequired then a time gate(s) would be placed immediately behind thecapture zone or zones. This use of the time gate may arise in caseswhere poor efficiency of binding of the component in the reactionmixture to the capture zone of the diagnostic element would prevail.

Another application of the time gate involves the placement of a timegate on a surface which is not part of a capillary space. For example,the time gate can be placed on a hydrophilic surface, which alonewithout a capillary space will allow liquids to move. This is generallythe case when a substantial volume of liquid is placed on a surface andit spreads because of surface tension and because of the hydrostaticpressure of the liquid pushing the meniscus outwardly. The time gatethen would function to delay the advance of the fluid front because thehydrostatic nature of the surface of the time gate would stop themovement of liquid. As the meniscus of the advancing liquid touches thetime gate, the component or components in the liquid binds to the timegate to create a sufficiently hydrophilic surface for a continuedadvance of the liquid on the surface.

Yet another embodiment of the time gate involves the positioning of atime gate prior to a membrane which is used to capture a conjugate orreceptor. In yet another embodiment of the time gate, the time gate canbe composed of hydrophobic surfaces in a membrane. In those cases, thehydrophobic membrane is positioned prior to the portion of membranewhich capture the conjugate or receptor and may be positioned after areaction chamber or a portion of membrane where reagents of the assayare placed or embedded and where the reagents incubate for a definedperiod of time. The time gate in the membrane can be formed byapplication of raw latex particle sin the membrane at an appropriatesolids concentration ranging from about 0.01% to 10%. The size of thelatex particles should be slightly less than the pore size of themembrane so that the latex becomes imbedded within the membrane. Thedensity of latex within the membrane at the time gate should be uniformso that the reaction mixture does not circumvent the time gate. Forexample, the latex size used to create a time gate for a membrane with apore size of 1 μm can range between 0.05 and 0.02 μm. Since thedistribution of pore sizes in membranes varies widely, the actual sizeof latex used must be arrived at by experimentation. The hydrophobicnature of the membrane used for the time gate can also be formed byplasma treatment or by treatment of the membrane with hydrophobicchemicals or polymers that adsorb to the membrane. One skilled in theart can appreciate that the teachings described herein of the inventivefeatures of the time gate can be utilized to design time gates in avariety of diagnostic devices which utilize membranes. That is, devicesdescribed, for example, in U.S. Pat. Nos. 4,435,504, 4,727,019,4,857,453, 4,877,586 and 4,916,056, and . . . hereby incorporated byreference, can incorporate a time gate, for example, prior to themembrane or in the membrane which captures the conjugate or receptor.

Optional Reagent Chambers

Referring to FIGS. 1b and 1c, the optional reagent chamber 17 is usefulfor the introduction of reagents into the assay process. In general, theoptional reagent chamber 17 may be in direct fluid contact with thesample addition reservoir 2 via a sample reaction barrier 3 or a portthe reaction chamber 4 or the diagnostic element 6, via a samplereaction barrier 3 or a port. For example, FIG. 1b shows the optionalreagent chamber 17 in direct fluid contact with the reaction chamber 4.The flow of the introduced reagent may be controlled by a time gate 5aand fingers 16 can aide in the movement of reagents into the reactionchamber 4. Referring now to FIG. 1c, for example, if a sequentialaddition immunoassay was to be performed by the device then 2 time gates5 and 5a would and fingers 16 can aid in the movement of reagents intothe reaction chamber 4. Referring now to FIG. 1c, for example, if asequential addition immunoassay was to be performed by the device then 2time gates 5 and 5a would allow 2 sequential incubation steps to beperformed in the optional reagent chamber 17 and then in the reactionchamber 4 by the device prior to the movement of the reaction mixtureonto the diagnostic element 6. That is, sample would be applied to thesample addition reservoir 2 through the sample addition zone 1 and thesample flows over the sample reaction barrier 3 and into the optionalreagent chamber 17 by the aid of fingers 16 where the first set ofreactions would occur. The time gate 5a, after the appropriate amount oftime, would allow the reagents to flow over the sample reaction barrier3a and into the reaction chamber 4 by the aid of fingers 16a where thenext set of reactions would take place. After the appropriate amount oftime, the time gate 5 allows the flow of reaction mixture onto thediagnostic element 6.

Fluid Control Means

Referring to FIG. 1d, the optional fluid control means 18 is designed tocontrol the flow of the reaction mixture in the device. Morespecifically, the optional fluid control means 18 causes the volume ofthe reaction mixture to flow over the capture zone of the diagnosticelement 6 at a rate which allows for an optimum capture of reagents ontothe capture zone. After the volume of the reaction mixture flows overthe capture zone the rate of flow of the excess reagents maybeincreased. The differential rate of flow of the reagents in the deviceis achieved by designing a gap 18 between the surfaces of the capillaryspace 19 of the diagnostic element 6. The size of the gap 18 is largerthan the capillary space 19 of the diagnostic element 6. The gap 18generally follows the capture zone or the zone where the rate of flow isrequired to be decreased. The gap 18 in the diagnostic element 6 thushas an associated volume. The volume of the gap 18 is filled with thereaction mixture by capillary action as it moves through the device.Since the gap 18 after the capture zone is greater than the capillaryspace 19 of the diagnostic element 6 a drop in capillary pressure at thebeginning of the gap 18 results in a decrease in the rate of flow of thereaction mixture into the gap 18 and therefore a decrease in the rate offlow of the reaction mixture over the capture zone. Varying the size ofthe gap 18 changes the capillarity in the gap and thus the flow of thereaction mixture over the capture zone. In the case of immunoassaysrequiring a wash step to remove unbound reagents from the diagnosticelement 6, it is generally desired that the rate of flow of the washsolution over the diagnostic element 6 is faster than the rate of flowof the reaction mixture over the diagnostic element 6 because thisdecreases the time of the assay. The shape of the gap can take manyforms. As shown in FIG. 1d, the gap has square corners, however, the gapcan be shaped as a trapezoid or triangle which would change the rate offlow of the reaction mixture while flowing into the gap. One skilled inthe art can also appreciate that for certain immunoassays a wash step isnot required.

The control of the rate of flow of the reagents in the device can alsobe used to allow chemical reactions to take place in one zone of thedevice before the reagents move to another area of the device where theextend of reaction of the reagents is monitored or where furtherreaction may take place. For example, several fluid control means couldbe incorporated into a device for use in immunoassays where a sequentialaddition and incubation of reagents is necessary. That is, the samplecomes in contact with the first reagents and the time for the reactionof the sample and first reagents is controlled by a first gap. When thefirst gap is filled with fluid, the reaction mixture continues to thesecond reagents at which time an additional chemical reaction cansubsequently take place. The time required for completion of this secondreaction can also be controlled by a second gap before further flow ofthe reaction mixture along the diagnostic element. Chemical andbiochemical reactions also take place in the volume of the gap, forexample, by immobilizing reagents in the gap.

Diagnostic Element

Referring to FIGS. 1 and 2, the diagnostic element 6 is formed byopposing surfaces which are a capillary distance apart through which thereaction mixture flows and on which are placed one or more capturezones. The capture zones are comprised of reagents, such as receptors,or devices, such as biosensors which bind or react with one or morecomponents from the reaction mixture. The binding of the reagents fromthe reaction mixture to the capture zones of the diagnostic element 6 isrelated to the presence or amount of target ligand in the sample. One ormore receptors or biosensors can be placed on the diagnostic element 6to measure the presence or amount of one or more target ligands. Thereceptors or biosensors can be placed in discrete zones on thediagnostic element 6 or they can be distributed homogeneously orheterogeneously over the surface. Receptors or other chemical reagents,for example, a receptor against the signal generator can also beimmobilized on the diagnostic element 6 to verify to the user that thereagents of the reaction mixture are viable and that the reactionmixture passed through the zones of the receptors or biosensors. Asingle receptor or biosensor can be placed over the majority of thediagnostic element 6 such that as the reaction mixture flows through thediagnostic element 6 the components from the reaction mixture bind tothe surface of the diagnostic element 6 in a chromatographic fashion.Thus, the distance which the component of the reaction mixture bindswould be related to the concentration of the target ligand in thesample. The reagents, such as receptors, are immobilized on the surfaceof the diagnostic element 6 through covalent bonds or throughadsorption. A preferred embodiment is to immobilize receptor coatedlatex particles, for example of diameters ranging from about 0.1 μm to 5μm. In addition, particles termed "nanoparticles" can also be coatedwith receptor and the resulting nanoparticles can be immobilized to thediagnostic element through adsorption or covalent bonds. Nanoparticlesare generally composed of silica, zirconia, alumina, titania, ceria,metal sols, and polystyrene and the like and the particle sizes rangefrom about 1 nm to 100 nm. The benefit of using nanoparticles is thatthe surface area of the protein coating the nanoparticle as a functionof the solids content is dramatically enhanced relative to larger latexparticles.

The surfaces of the diagnostic element 6 would allow the receptor coatednanoparticles or latex particles to bind to the diagnostic element 6. Ina preferred embodiment, the receptors bind to the surface of thediagnostic element through electrostatic, hydrogen bonding and/orhydrophobic interactions. Electrostatic, hydrogen bonding andhydrophobic interactions are discussed, for example, in Biochemistry 20,3096 (1981) and Biochemistry 29, 7133 (1990). For example, thediagnostic element 6 can be treated with a plasma to generate carboxylicacid groups on the surface. The receptor coated latex particles arepreferably applied to the diagnostic element 6 in a low salt solution,for example, 1-20 mM, and at a pH which is below the isoelectric pointof the receptor. Thus, the negative character of the carboxylic acidgroups on the diagnostic element 6 and the positive charge character ofthe receptor latex will result in enhanced electrostatic stabilizationof the latex on the diagnostic element 6. In another preferredembodiment, latex particles or nanoparticles, which may be coated withreceptor or may compose a time gate, are entrapped on a non-absorbentsurface. The microstructure of the non-absorbent surface is textured sothat the particles are entrapped on the surface or in the layers of themicrostructure, forming what is generally referred to as a"nanocomposite." Magnetic fields may also be used to immobilizeparticles which are attracted by the magnetic field. These types ofsurfaces, generally termed "nanostructured materials" are described, forexample, in Chemical and Engineering News 70, 18-24 (1992), herebyincorporated by reference.

In an additional embodiment of the diagnostic element, now referring toFIG. 5, the diagnostic element 6 is a cylindrical surface which may becomposed of grooves. When the diagnostic element is composed of grooves,the grooves generally run perpendicular to the flow of the reactionmixture. A capillary space is formed around the diagnostic element by around tube which is generally clear; thus, the surface of the diagnosticelement and the opposing surface of the tube are a capillary distanceapart. The capillary formed allows the flow of the reaction mixture overthe round diagnostic element 6. Generally, the reaction mixture wouldtravel up against gravity or down with gravity through the cylindricalcapillary space. The capture zones of the round diagnostic element 6 canbe placed in discrete zones or over the entire length of the diagnosticelement 6. The capture zones may also circle the diameter of thediagnostic element 6 or may be applied to only a radius of thediagnostic element 6. The reaction mixture may be delivered to thediagnostic element 6 through the tube 8. Furthermore, the cylindricalvolume of the tube 8 may be used as a reaction chamber 4 and a discshaped sample reaction barrier 3 with grooves on its perimeter may alsobe inserted to form the reaction chamber 4 and the sample additionreservoir 2. From this discussion, now referring to FIGS. 1 and 2, oneskilled in the art can also appreciate that the flat diagnostic element6 may also be curved such that the curvature is a radius of a circle.

One skilled in the art can appreciate that various means can be used forthe detection of signal at the capture zone of the diagnostic element.In the case of the use of biosensors, such as, for example, apiezoelectric crystal, the piezoelectric crystal onto which would beimmobilized a receptor, would be the capture zone and the responsegenerated by binding target ligand would be generally reflected by anelectrical signal. Other types of detection means include, but are notlimited to visual and instrumental means, such as spectrophotometric andreflectance methods. The inventive features of the diagnostic elementdescribed herein allows for improved capture efficiencies on surfacesover which a reaction mixture flows and that various means for detectionmay be used by one skilled in the art.

The surfaces of the capillaries in the device are generally hydrophilicto allow flow of the sample and reaction mixture through the device. Ina preferred embodiment the surface opposing the diagnostic element 6 ishydrophobic such that the reaction mixture repels this surface. Therepulsion of reaction mixture to the surface opposing the diagnosticelement 6 forces the reaction mixture, and particularly the proteinconjugates, to the surface where capture occurs, thus improving thecapture efficiency of the components of the reaction mixture to thecapture zone. The hydrophobic surfaces opposing the diagnostic elementcan have a tendency to become hydrophilic as the reaction mixtureprogresses through the diagnostic element because various componentswhich may be present endogenously or exogenously in the sample orreaction mixture, such as, for example, proteins or polymers, bind tothe hydrophobic surface. A preferred hydrophobic surface opposing thediagnostic element can be composed of teflon. It is well known to thoseskilled in the art that teflon surfaces bind proteins poorly. Thus, theteflon surface opposing the diagnostic element would not become ashydrophilic as would surfaces composed of, for example, polystyrene,polyacrylate, polycarbonate and the like, when the reaction mixtureflows through the diagnostic element.

In another preferred embodiment, the diagnostic element 6 is hydrophilicbut the areas adjacent to the diagnostic element 6 are hydrophobic, suchthat the reagents of the assay are directed through only the hydrophilicregions of the diagnostic element. One skilled in the art will recognizethat various techniques may be used to define a hydrophilic diagnosticelement or zone, such as plasma treatment of hydrophobic surfaces usingmasks which shield the surfaces, except for the diagnostic element, fromthe treatment or by application of hydrophobic adhesives to hydrophilicsurfaces to define a diagnostic element or by the use of viscoushydrophobic compounds, such as an oil or a grease. In another preferredembodiment, the capillary of the diagnostic element can be formed byultrasonic welding. The boundaries of the diagnostic element aredictated by the energy directors which are used to form the sonicatedweld.

The surfaces of the diagnostic element 6 or of the other components ofthe device may be smooth or grooved or grooved and smooth. Varioustextured surfaces may also be employed, alone or in combination withsmooth or grooved surfaces. For example, surfaces composed of posts,grooves, pyramids and the like, referred to as protrusions, or holes,slots, waffled patterns and the like, referred to as depressions may beutilized. The textured geometries may be ordered in rows, staggered ortotally random and different geometries may be combined to yield thedesired surface characteristics. The depressions or the protrusions ofthe textured geometries can range from about 1 nm to 0.5 mm andpreferably from about 10 nm to 0.3 mm. The distance between the variousdepressions and protrusions can range from about 1 nm to 0.5 mm andpreferably from about 2 nm to 0.3 mm.

In a preferred mode as shown in FIGS. 1 and 2, one surface of thediagnostic element 6 is grooved and the grooves are perpendicular to theflow of the reaction mixture and the opposing surface is smooth. Inanother embodiment, one surface of the diagnostic element 6 is groovedat the capture zone and the areas adjacent to the capture zone aresmooth. The opposing surface of the diagnostic element 6 may be smoothor may be grooved, for example, the grooves of each surface intermesh.The positioning of the grooves of the diagnostic element perpendicularto the flow of the reaction mixture is beneficial in that the flow ofthe reaction mixture through the diagnostic element 6 occurs in anorganized manner with a distinct, straight front dictated by the groovesin the capillary space. In addition, when one surface is in closeproximity, for example 1 μm to 100 μm, to the peaks of the grooves thenthe capture efficiency of the components from the reaction mixture canbe enhanced. The enhancement of capture efficiency at the capture zonesin grooved diagnostic elements as compared to smooth surface elementsmay be related to the movement of the reaction mixture in the capillaryspace; that is, in the case of the grooved surface the reaction mixtureis forced to move over the peak of the groove and into the trough of thenext groove. Thus, a finer grooved surface, that is, more grooves percm, would provide a better capture efficiency than a coarser groovedsurface. The reaction mixture is thus driven closer to the surface ofthe grooved diagnostic element than it would be if both surfaces weresmooth. Also, the close proximity of the surfaces decreases the volumeof the bulk reaction mixture above the grooved surface of the diagnosticelement and therefore decreases the diffusion distance of the componentswhich bind to the diagnostic element. The proximity of the surfaces ofthe diagnostic element should minimize the volume of reaction mixture inthe diagnostic element at the capture zone without blocking thecapillary flow through the element. The capture of, for example, thecomplex of target ligand: Ligand receptor conjugate at the capture zonecan approach 100% efficiency if the proximity of the surfaces isoptimized. The capture of nearly all of the ligand receptor conjugatewhich is bound by target ligand is most desired because a greatersensitivity of the assay as a function of sample volume can be achieved.Other advantages of improved capture efficiency are that less reagentsare used because the sample volume is decreased, the assay device can beminiaturized because of the smaller sample volume and thereproducibility of the assay result will be improved because changes inthe rate of flow of the reaction mixture through the capture zones willhave less or no effect on the capture of the labelled conjugates.

The capillary space can be defined by a variety of ways, for example,machining the surfaces to the appropriate tolerances or using shimsbetween the surfaces. In a preferred embodiment, ultrasonic welding ofthe surfaces defines the capillary. In this case, the capillary space isdefined by the energy directors and the distance between the surfaces isa function of the size of the energy director, the welding energy, thetime of energy application and the pressure applied during welding. Thesurfaces of the diagnostic element can be parallel or non-parallel. Inthe latter case, the flow rate of the reagents through the diagnosticelement will not be uniform throughout the length. A preferredembodiment is to maintain the surfaces of the diagnostic elementapproximately parallel. The surfaces of the diagnostic element can bemade from materials, such as plastics which are capable of being milledor injection molded, for example, polystyrene, polycarbonate,polyacrylate and the like or from surfaces of copper, silver and goldfilms upon which are adsorbed various long chain alkanethiols asdescribed in J.Am.Chem.Soc. 1992, 114, 1990-1995 and the referencestherein. In this latter example, the thiol groups which are orientedoutward can be used to covalently immobilize proteins, receptors orvarious molecules or biomolecules which have attached maleimide or alkylhalide groups and which are used to bind components from the reactionmixture for determining the presence or amount of the target ligand.

Referring to FIGS. 3a and 3b, the zones of immobilization of one or morereceptors or the placement of biosensors at the capture zone 17 on thediagnostic element 6 can take many forms. For example, if the targetligand is very low in concentration in the sample then one would desirethat all of the reaction mixture pass over the zone of immobilizedreceptor or biosensor to obtain the best signal from the given volume ofreaction mixture. In this case, the placement of the reagents orbiosensors on the diagnostic element 6 at the capture zones 17 could,for example, resemble that shown in FIG. 3a. If the target ligand in thesample is high in concentration and the sensitivity of the analyticalmethod is not an issue then the placement of the receptors or biosensorsat the capture zones 17 could, for example, resemble that in FIG. 3b.One skilled in the art can appreciate that the placement of receptors orbiosensors on the diagnostic element is a function of the sensitivityrequirements of the analytical method.

One or more diagnostic elements can comprise a device. The reactionmixture may be applied to a device with multiple diagnostic elements. Inaddition, the sample may be applied to the device and then separatedinto different reaction chambers, each with separate diagnosticelements. The capture zone can be various geometrical symbols or lettersto denote a code when the sample is positive or negative for the targetligand. One skilled in the art will recognize the useful combinations ofthe elements of this invention.

The diagnostic element can also be configured to perform asemi-quantitative or quantitative assay, as for example, is described inClinical Chemistry (1993) 39, 619-624, herein referred to by referenceonly. This format utilizes a competitive binding of antigen and antigenlabel along a solid phase membrane. The improvement is that the use ofthe diagnostic element described herein for the above cited method wouldrequire a smaller sample volume and improved binding efficiency to thesolid phase surface.

Diagnostic Elements Other Than Capillaries

The inventive teachings described herein of the adsorbtion of proteins,particularly receptors to plastic surfaces, can be utilized foradsorbtion of receptors to many plastic surfaces which are not a part ofa capillary. Nanoparticles and latex particles coated with receptors canalso be applied to surfaces of many types of immunoassay devices, as forexample, to "dipsticks." Dipsticks are generally used as a solid phaseonto which are bound, as a result of the assay process, for example, theligand receptor conjugate. Dipsticks generally incorporate membranes;however, a disadvantage in the use of membranes in dipsticks is thedifficulty in washing the unbound ligand receptor from the membrane.Thus, an improvement in the use of dipsticks is to immobilize receptorcoated latex or nanoparticles directly onto a plastic surface of thedipstick. The removal of unbound ligand conjugate from the plasticsurface is thus more efficient than removal from a membrane.

Used Reagent Reservoir

Referring to FIGS. 1 and 2, the used reagent reservoir 7 receives thereaction mixture, other reagents and excess sample from the diagnosticelement 6. The volume of the used reagent reservoir 7 is at least thevolume of the sample and extra reagents which are added to or are in thedevice. The used reagent reservoir 7 can take many forms using anabsorbent, such as a bibulous material of nitrocellulose, porouspolyethylene or polypropylene and the like or the used reagent reservoircan be comprised of a series of capillary grooves. In the case ofgrooves in the used reagent reservoir 7, the capillary grooves can bedesigned to have different capillary pressures to pull the reagentsthrough the device or to allow the reagents to be received without acapillary pull and prevent the reagents from flowing backwards throughthe device. The size and quantity of the grooved capillaries determinethe volume and capillarity of the used reagent reservoir 7. In apreferred embodiment, as shown in FIG. 4, the fingers 52 at the end ofthe diagnostic element 6 are in fluid contact with a capillary space 55and the capillary space 55 is in fluid contact with a grooved ortextured capillary space 56. The depth of the grooves or texturedsurface can be, for example, about 0.1 mm to 0.6 mm, preferably about0.3 mm to 0.5 mm and the density can range from about 5 to 75 groovesper cm and preferably about 10 to 50 grooves per cm. Referring to FIG.4, the reagents of the device move to the fingers 52 at the end of thediagnostic element 51 and into the capillary channel 55. The reagentseither partially or completely fill the capillary space 55 and then comein contact with the grooved or textured surface 56. The width of thecapillary space 55 is generally about 1 mm to 3 mm and the depth isgenerally about 0.1 mm to 2 mm. The length of the capillary space 55should be sufficient to be in fluid contact with the grooved or texturedsurface 56. The grooved or textured surface 56 partially or completelypulls the reagents from the capillary channel 55 depending on the rateof delivery of the reagents into the capillary space 55 from thediagnostic element 51. When the flow of reagents is complete in thedevice, the grooved or textured surface 56 has greater capillarity thanthe capillary channel 55 and the reagents are removed from the capillarychannel 55 by the grooved or textured surface 56. In addition, thereverse flow of the reagents from the grooved or textured surface is notpreferred because the capillarity in the grooved or textured surface 56holds the reagents and prevents their backward flow. One skilled in theart can recognize from these inventive features that the arrangement ofgrooves or a used reagent reservoir within the device can be adapted toa variety of desired objectives.

The Description of the One-Step Assay Device

The elements of the device which have been described individually can beassembled in various ways to achieve the desired function. The term"one-step" implies that one manual action is required to achieve theassay result, for example, adding sample to the device is one step. Inthe case of the device performing a one-step assay which involves both atimed incubation of reagents and a wash step, the wash solution isexcess sample and the assay device is built with the elements in fluidcommunication using the sample addition reservoir, the sample-reactionbarrier, the reaction chamber, the time gate, the diagnostic element andthe used reagent reservoir as depicted in FIG. 1. The devices aregenerally about 3 cm to 10 cm in length, 1 cm to 4 cm in width and about2 mm to 15 mm thick. Typically, a top member with smooth surfaces isplaced onto a bottom member which has a surface onto which are built theelements stated above. The relationship of the elements are as depictedin FIG. 1. The reagents required for performing the assay areimmobilized or placed in the respective elements. The surfaces arebrought together, a capillary distance apart, and in doing so, theregions of the sample addition reservoir, the sample reaction barrier,the reaction chamber, the time gate, the diagnostic element, the gap andthe used reagent reservoir are all formed and are capable of functioningtogether. Also, the surfaces are brought together such that the opposingsurfaces touch to form and seal the sample addition reservoir, thereaction chamber, and the used reagent reservoir.

When performing a qualitative, non-competitive assay on one or moretarget ligands, the signal producing reagents, which could include, forexample, a receptor specific for the target ligand adsorbed to acolloidal metal, such as a gold or selenium sol, are placed on thesample reaction barrier or in the reaction chamber in dried orlyophilized form. Another receptor for each target ligand is immobilizedonto the surface of the diagnostic element at the capture zone. The timegate is positioned generally on the diagnostic element between thereaction chamber and the capture zones by the placement of, for example,a surfactant-free polystyrene suspension onto the device in an amountwhich dictates the desired incubation time. The incubation time isusually the amount of time for the reactions to come to substantialequilibrium binding. The assay is then performed by addition of sampleto the sample addition reservoir of the device. The sample moves overthe sample-reaction barrier, into the reaction chamber by the aid of thefingers and dissolves the reagents in the reaction chamber to form thereaction mixture. The reaction mixture incubates for the amount of timedictated by the time gate. The excess sample remaining in the sampleaddition reservoir and reaction mixture in the reaction chamber are influid communication but are not in substantial chemical communicationbecause of the sample-reaction barrier. Thus, the reaction chamberdefines the volume of the reaction mixture. The reaction mixture thenmoves past the time gate and onto the diagnostic element and over thecapture zones. The complex of receptor conjugate and target ligandformed in the reaction mixture binds to the respective receptor at thecapture zone as the reaction mixture flows over the capture zones. Thereaction mixture may also flow over a positive control zone, which canbe for example, an immobilized receptor to the signal developmentelement. As the reaction mixture flows through the diagnostic elementand into the used reagent reservoir by the aid of the fingers, theexcess sample flows behind the reaction mixture and generally does notsubstantially mix with the reaction mixture. The excess sample movesonto the diagnostic element and removes the receptor conjugate which didnot bind to the capture zone. When sufficient excess sample washes thediagnostic element, the signal at the capture zones can be interpretedvisually or instrumentally. Referring to FIG. 1d, in a preferred mode ofthe above description, the reaction mixture moves onto the diagnosticelement 6, over the capture zone or zones and then the reaction mixtureproceeds into a capillary gap 18. The capillary gap 18 generally hasless capillarity than that of the diagnostic element 6. the capillaryspace 19 of the diagnostic element 6 is generally smaller than thecapillary space of the gap 18. The volume of the capillary gap 18generally approximates the volume of the reaction mixture such that thecapillary gap 18 fills slowly with the reaction mixture and once filled,the capillarity of the remaining portion of the diagnostic element 6 orused reagent reservoir is greater than the capillarity of the gap 18resulting in an increased rate of flow to wash the diagnostic element 6.As one skilled in the art can appreciate, the gap 18 can be formed inthe top member 8 or in the bottom member 9 or a combination of bothmembers 8 and 9.

In the case of the device performing a one-step assay which does notinvolve a timed incubation step but does involve a wash step in whichthe wash solution is excess sample, the assay device is built with theelements in fluid communication using the sample addition reservoir, thesample-reaction barrier, the reaction chamber, the diagnostic elementand the used reagent reservoir. The assay reagents are used as describedabove for the non-competitive qualitative assay. The assay devicewithout the time gate allows the reaction mixture to flow onto thediagnostic element without an extended incubation time. The capillaryflow of the reaction mixture and the excess sample are as describedabove.

The optional reagent chamber is incorporated into the device in the caseof the device performing a one-step assay with the introduction of anadditional assay reagent into or after the reaction mixture or theintroduction of a wash solution which flows behind the reaction mixturethrough the device. The optional reagent chamber may be in fluid contactwith any element of the device and is generally in fluid contact withthe reaction chamber. When in fluid contact with, for example, thereaction chamber, the optional reagent chamber and the reaction chambermay be separated by a time gate. Various reagents may be dried orlyophilized in the optional reagent chamber, such as detergents for awashing step or reagents which are sequentially provided to thediagnostic element after the reaction mixture.

In the case of performing one-step, non-competitive, quantitative assaysthe reagents as described above for the non-competitive, qualitativeassay may apply. The device is comprised of the elements, sampleaddition reservoir, sample-addition barrier, reaction chamber, timegate, diagnostic element and used reagent reservoir. In this case, thecapture zone of the diagnostic element is generally the entirediagnostic element. That is, the capture zone is a length of thediagnostic element onto which the receptor conjugate binds. The receptorconjugate binds along the length of the capture zone in proportion tothe amount of target ligand in the sample. The device of the presentinvention is preferred for this quantitative assay because of the highefficiency of capture of the reagents, for example, the binding of acomplex of target ligand and receptor conjugate to an immobilizedreceptor to the target ligand on the capture zone, and because themovement of the reaction mixture over the diagnostic element proceedswith a sharp front. The receptors on the capture zone sequentiallybecome saturated with the complex of target ligand and receptorconjugate as the reaction mixture moves over the length of the capturezone. The length of the diagnostic element containing bound conjugatethen determines the concentration of the target ligand. Those skilled inthe art will recognize the format of this type of immunoassay as aquantitative immunochromatographic assay as discussed in U.S. Pat. Nos.4,883,688 and 4,945,205, hereby incorporated by reference.

In the case of the device performing a one-step, qualitative,competitive assay which involves both a timed incubation or reagents anda wash step and the wash solution is excess sample, the assay device isbuilt with the elements in fluid communication using the sample additionreservoir, the sample-reaction barrier, the reaction chamber, the timegate, the diagnostic element and the used reagent reservoir. Whenperforming a qualitative competitive assay on one or more targetligands, the conjugate is composed of, for example, a ligand analoguecoupled to signal development element, such as a gold or selenium sol.The conjugate and receptor for each target ligand are place din thereaction chamber in dried or lyophilized form, for example, in amountswhich are taught by U.S. Pat. Nos. 5,028,535 and 5,089,391, herebyincorporated by reference. Another receptor for each target ligand isimmobilized onto the surface of the diagnostic element at the capturezone. The time gate is positioned generally on the diagnostic elementbetween the reaction chamber and the capture zones as describedpreviously. The incubation time is usually the amount of time for thereactions to come to substantial equilibrium binding. The assay is thenperformed by addition of sample to the device. The sample moves over thesample-reaction barrier and into the reaction chamber, dissolves thereagents to form the reaction mixture and incubates for the timedictated by the time gate. The excess sample and reaction mixture are influid communication but not in substantial chemical communicationbecause of the sample-reaction barrier. The reaction mixture then movesonto the diagnostic element and over the capture zones. The ligandanalogue conjugate binds to the respective receptor or receptors at thecapture zone or zones. As the reaction mixture flows over the diagnosticelement and into the used reagent reservoir, the excess sample flowsbehind the reaction mixture and generally does not substantially mixwith the reaction mixture. The excess sample moves onto the diagnosticelement and removes conjugates which do not bind to the capture zone orzones. When sufficient excess sample washes the diagnostic element theresults at the capture zones can be interpreted visually orinstrumentally. In a preferred mode of the above invention, the reactionmixture moves onto the diagnostic element, over the capture zone orzones and then the reaction mixture proceeds into a capillary gap. Thecapillary gap has less capillarity than that of the diagnostic element.The volume of the capillary gap generally approximates the volume of thereaction mixture such that the capillary gap fills slowly with thereaction mixture and once filled, the capillarity of the remainingportion of the diagnostic element or used reagent reservoir is greaterresulting in an increased rate of flow of excess sample to wash thediagnostic element.

In another aspect of the one-step, competitive assay, the reactionmixture is composed of ligand analogue-ligand complement conjugate toeach target ligand and receptors adsorbed to latex particles withdiameters of, for example, 0.1 μm to 5 μm to each target ligand, inappropriate amounts, for example, as taught by U.S. Pat. Nos. 5,028,535and 5,089,391. The ligand complement on the conjugate can be anychemical or biochemical which does not bind to the receptors for thetarget ligands. The assay is begun by addition of sample to the device.Sample fills the reaction chamber and is incubated for a time whichallows the reagents to come to substantial equilibrium binding. Thereaction mixture flows over the time gate and onto or into a filterelement to prevent ligand analogue-ligand complement conjugates whichhave bound to their respective receptor latexes from passing onto thediagnostic element. Typical filter elements can be composed ofnitrocellulose, cellulose, nylon, and porous polypropylene andpolyethylene and the like. Thus, only the ligand analogue-ligandcomplements conjugate which were not bound by the receptor latex willpass onto the diagnostic element. The receptor to the ligand complementof the conjugate is immobilized on the diagnostic element at the capturezone and bind the conjugate. A wash step may not be required because thefilter removes the conjugate bound to latex; however, the excess sampleor a wash solution from the optional reagent chamber may be used to washthe diagnostic element.

In the case of a one-step quantitative, competitive assay, the receptorto the ligand analogue conjugate or the ligand complement of theconjugate is immobilized onto the diagnostic element as describedpreviously for the one-step quantitative, non-competitive assay. Thus,the concentration of the target ligand in the sample is visualized bythe distance of migration on the diagnostic element of the conjugate. Inanother mode, a quantitative assay could be performed by the binding ofthe labelled conjugate, for example, the ligand analogue-ligandcomplement conjugate, to sequential, discrete capture zones of receptoron the diagnostic element. The quantitative result is achieved by thedepletion of the conjugate as the reaction mixture flows through thecapture zones of the diagnostic element.

The Device as a Diagnostic Element

The diagnostic element of the device can be utilized with a sampleaddition means to perform a separation step of bound and unboundconjugates. An example of this type of device which has a sampleaddition means, a diagnostic element and a used reagent reservoir isdepicted in FIG. 2. For example, in the case of a non-competitive assay,at least one receptor conjugate is incubated with sample which issuspected of containing at least one target ligand in a suitable vesseland this reaction mixture is applied to the sample addition zone of thedevice. The reaction mixture then flows onto the diagnostic element andover the capture zone of, for example, immobilized receptor to thetarget ligand. When target ligand is present in the sample, the targetligand-receptor conjugate complex binds to the receptor on the capturezone. If the signal development element is an enzyme, then either asubstrate for the enzyme which produces a visual color or a washsolution followed by a substrate is next added to the device. Excessreagents flow to the used reagent reservoir. The presence or amount ofeach target ligand in the sample is then determined either visually orinstrumentally.

In the case of a competitive immunoassay, for example as taught by U.S.Pat. Nos. 5,028,535 and 5,089,391, herein incorporated by reference, thediagnostic element may be used to separate bound and unbound ligandanalogue conjugates such that the unbound ligand analogue conjugatesbind to the receptors of the diagnostic element in proportion to thepresence or amount of target ligand in the sample.

One skilled in the art can appreciate that all formats of immunoassaysor gene probe assays which require a separation step of free and boundconjugates or the separation of free of bound reagents whichsubsequently leads to the ability to detect a signal can utilize theinventive features of the diagnostic element. One skilled in the art canalso recognize that the inventive elements of this invention, namely,the fingers, the sample reaction barrier, the reaction chamber, the timegate, the diagnostic element, the fluid control means and the usedreagent reservoir can be used separately or in various combinations andin conjunction with other devices not described here. For example, thesample reaction barrier with fingers and the reaction chamber can beused in conjunction with devices incorporating porous members, such asmembranes to deliver precise volumes of reagents to the porous member.The time gate can also be incorporated into the aforementioned devicesor the time gate may be used alone in conjunction with devicesincorporating porous members. The fluid control means can also be usedin devices incorporating porous members to control the rate of flow ofreagents through the porous member.

Experimental Procedures EXAMPLE 1 Preparation of Anti-βhCGAntibody-Colloidal Gold Conjugate

Colloidal gold with an average diameter of 45 nm was prepared accordingto the method of Frens, Nature, Physical Sciences, 241, 20 (1973). Thecolloidal gold conjugate was prepared by first adding 5.6 ml of 0.1 Mpotassium phosphate, pH 7.58, dropwise with rapid stirring to 50 ml ofcolloidal gold. Anti β-subunit monoclonal antibody to hCG (AppliedBiotech, San Diego, Calif.; 1 ml of 4.79 mg/ml in phosphate bufferedsaline, 0.02% sodium azide, pH 7) was added in a bolus to the colloidalgold with rapid stirring. After complete mixing the stirring was stoppedand the solution was incubated at room temperature for 1 h. Polyethyleneglycol (average molecular weight=20,000) was added (0.58 ml) as a 1%solution to the colloidal gold solution and the solution was mixed. Thecolloidal gold solution was subjected to centrifugation at 27,000 g and5° C. for 20 min. The supernatant was removed and each pellet was washedtwice by resuspension and centrifugation with 35 ml of 10 mM potassiumphosphate, 2 mM potassium borate, 0.01% polyethylene glycol (averagemolecular weight=20,000), pH 7. After the final centrifugation, thepellet was resuspended in 0.5 ml of the wash buffer. The gold conjugatewas diluted for the assay of hCG into a buffered solution containing 10mg/ml bovine serum albumin at pH 8.

EXAMPLE 2 Preparation of Anti-αhCG Antibody Latex

Surfactant-free polystyrene particles (Interfacial Dynamics Corp.,Portland, Oreg.; 0.106 ml of 9.4% solids, 0.4 μm) was added whilevortexing to anti α-subunit hCG monoclonal antibody (Applied Biotech,Dan Diego, Calif.; 0.89 ml of 6.3 mg/ml in 0.1 M 2-(N-morpholino) ethanesulfonic acid, (MES), pH 5.5) and the suspension was incubated at roomtemperature for 15 min. The suspension was subjected to centrifugationto pellet the latex particles. The pellet was washed three times bycentrifugation and resuspension of the pellet with 10 mM MES, 0.1 mg/mltrehalose, pH 5.5. The final pellet was resuspended in the wash bufferat a solids concentration of 1%.

EXAMPLE 3 Preparation of Goat Anti-Mouse Latex

Surfactant-free polystyrene particles (Interfacial Dynamics Corp.,Portland, Oreg.; 0.11 ml of 9.4% solids, 0.6 μm) were added whilevortexing to goat IgG antibody against mouse IgG (Jackson ImmunoResearchLaboratories, Inc.; 0.89 ml of 0.34 mg/ml in 0.1 M MES, pH 5) and thesuspension was incubated at 45° C. for 2 h. The suspension was subjectedto centrifugation to pellet the latex particles. The pellet was washedthree times by centrifugation and resuspension of the pellet with 10 mMMES, 0.2 mg/ml trehalose, pH 5.5. The final pellet was resuspended inthe wash buffer at a solids concentration of 1%.

EXAMPLE 4 Preparation of the One-Step Device for a Qualitative hCG Assay

A one-step device made of plastic was built having an 80 to 100 μlsample addition reservoir, a 20 μl reaction chamber and a 40 μl usedreagent reservoir. This device is designed for applying samples of about20 μl to 100 μl, but the reaction chamber is fixed at 20 μl. In caseswhere a larger reaction mixture volume is required for the desiredassay, then the reaction chamber would be increased to that volume andthe sample addition reservoir would be about 2 to 4 times the volume ofthe reaction chamber volume. The devices were plasma treated to graftfunctional groups which create a hydrophilic surface. Those skilled inthe art will recognize that the plasma treatment of plastic is performedin a controlled atmosphere of a specific gas in a high frequency field.The gas ionizes, generating free radicals which react with the surface.The sample addition reservoir was shaped as a trapezoid with dimensionsof 14 mm and 7 mm for the parallel sides and 7 mm for the other sideswith a depth of 0.49 mm. The sample addition reservoir was adjacent tothe sample reaction barrier. The sample-reaction barrier was 1.5 mm longand 7 mm wide including grooves running parallel to the flow of thesample at a density of 50 grooves per cm and a depth of 0.1 mm. In thecase of sample volumes larger than 20 to 80 μl, the width of thereaction barrier and thereby the reaction chamber could be increased toaccommodate the desired flow rate but the groove size or density couldremain as indicated. The fingers in the walls of the reaction chamberand the used reagent reservoir were 1 mm wide and 0.4 mm deep with 7fingers in each wall of the reaction chamber and the used reagentreservoir. The reaction chamber volume was 20 μl. The reaction chamberwas shaped as a trapezoid with dimensions of 7 mm and 3.5 mm for theparallel sides and 7.1 mm for the other sides with depths of 0.56 mm for20 μl reaction chambers. The diagnostic element was about 2.5 cm long, 2mm wide and 1 mm from the base of the device including grooves runningperpendicular to the flow of reaction mixture at a density of 100grooves per cm and a depth of 0.05 mm. In the case of a time gate on thediagnostic element, the time gate was positioned on the diagnosticelement immediately adjacent to the reaction chamber. The width of thediagnostic element could be increased to increase the flow of thereaction mixture to the desired rate past the capture zones. Theanti-αhCG antibody latex (1 μl) and the goat anti-mouse latex (1 μl)were applied to the diagnostic element of the devices approximately 1.5cm apart. The anti-βhCG antibody colloidal gold conjugate (10 μl) waspipetted into the trough of the reaction chamber. The devices wereplaced under vacuum for about 15 min. to dry the reagents. The usedreagent reservoir had the shape of a trapezoid with dimensions of 7 mmand 15 mm for the parallel sides and 8 mm for the other sides with adepth of 0.5 mm. Referring to FIG. 4, in a preferred (best mode)embodiment of the used reagent reservoir, the reaction mixture moved toa capillary space 55 (1.25 mm long, 27.5 mm wide and 0.48 mm deep) fromthe diagnostic element 6, aided by fingers 52 (1 mm wide and 0.4 mm deepwith 7 fingers), and then into a grooved capillary structure (13.6 mmlong, 25.4 mm wide, 0.61 mm deep with a density of 16 grooves per cm).The outer walls and the top surface of the walls of the sample additionreservoir and the reaction chamber had applied a thin coating of silicongrease to prevent the leakage of the reagents from the reservoir andchamber of the assembled device. The capillary spaces in the deviceswere then formed by placing a clear plastic polycarbonate sheet on topof the device. The plastic sheet was held to the opposing surface withbinder clips. The clear plastic sheet had a sample port above the sampleaddition reservoir for the introduction of sample.

EXAMPLE 5 Qualitative One-Step Assay for hCG

The devices described in Example 4 were used for the qualitativeone-step assay for hCG. The assay times for the devices without the timegates were about 5 to 10 min. A urine solution (60 μl) containing 0, 50,200 and 500 mIU hCG/ml was added to the sample reservoir of the devices.The sample moved into the reaction chamber, dissolved the colloidal goldconjugate and the reaction mixture moved onto the diagnostic elementover the anti-hCG latex and goat anti-mouse IgG latex capture zones. Thereaction mixture moved into the used reagent reservoir and the excesssample washed the diagnostic element. The color density of the capturezones for hCG was measured instrumentally using a Minolta Chroma MeterCR 241 at 540 nm. A red color was visible for samples containing hCG andnot visible for the sample without hCG at the capture zones for hCG. TheΔE* values for the 0, 50, 200 and 500 mIU/ml were 0, 7.78, 12.95 and20.96, respectively, and for the positive control (goat anti-mouse IgG)zones a distinctive red bar was observed with a ΔE* of about 35.

EXAMPLE 6 Qualitative One-Step Assay for hCG Using a Time Gate

Devices as described in Example 4 were prepared with the addition of thetime gate. The time gate was formed on the diagnostic element which isin contact with the reaction mixture in the reaction chamber. The timegate was prepared by adding 1 μl of 2% solids of surfactant-free,sulfated latex, 1.0 μm, (Interfacial Dynamics Corp., Portland, Oreg.).The other reagent latexes and gold conjugate were also added to thedevices and dried as described in Example 5. Clear plastic sheets wereplaced on the devices and sample (about 60 μl) containing 0, 50, 200 and500 mIU hCG/ml was added to the devices. The sample moved into thereaction chamber, dissolved the colloidal gold conjugate and thereaction mixture remained in the reaction chamber for about 8 to 10 min,whereas in devices without time gates the reaction mixture remained inthe reaction chamber for 5 sec to 15 sec. The proteinaceous componentsof the reaction mixture, which may be present in the sample and whichwas added as a component of the reaction mixture, namely, bovine serumalbumin, bound to the latex particles of the time gate and changed thehydrophobic surface of the time gate into a hydrophilic surface. Otherproteins, such as gelatin, serum albumins, immunoglobulins, enzymes andthe like and polypeptides and hydrophilic polymers will also function tobind to the hydrophobic zone. The gradual transformation of thehydrophobic surface of a hydrophilic surface, which resulted throughbinding of the proteinaceous components of the reaction mixture to thelatex particles allowed the reaction mixture to flow over the area ofthe time gate. In control experiments in which protein, namely bovineserum albumin, was not added to the reaction mixture, flow of thereaction mixture over the time gate and onto the diagnostic element didnot occur during the time (5 h) of the experiment. This controlexperiment showed that the urine sample alone did not contain sufficientprotein or components which bind to the applied latex of the time gateto allow a change in the hydrophobic character of the time gate. In theevent that the components in the sample should only be used to cause thetransformation of the hydrophobic time gate to a hydrophilic one for thereaction mixture to flow, then one would be required to lower the massand total surface area of the latex applied to the time gate to anextent which would allow flow of the reaction mixture over the time gatein an appropriate amount of time. The reaction mixture then moved ontothe diagnostic element over the anti-hCG latex and goat anti-mouse IgGlatex capture zones. The reaction mixture moved into the used reagentreservoir and the excess sample washed the diagnostic element. The colordensity of the capture zones for hCG was measured instrumentally using aMinolta Chroma Meter CR 241. A red color was visible for samplescontaining hCG and not visible for the sample without hCG at the capturezones for hCG. The ΔE* values for the 0, 50, 200 and 500 mIU/ml were 0,6.51, 13.14 and 18.19, respectively. A red color bar was visible at thegoat anti-mouse IgG capture zones of each device.

EXAMPLE 7 Qualitative One-Step Assay for hCG Using a Flow Control Means

Devices as described in Example 4 were prepared with the addition of theoptional flow control means. The optional flow control means or "gap"was placed behind the capture zone for hCG gold conjugate on thediagnostic element. The gap between the two surfaces was 0.38 mm, thelength of the gap was 13.2 mm and the width of the gap on the top memberwas 9 mm; however, the effective width of the gap was the width of thediagnostic element (2 mm). This gap volume above the diagnostic elementwas about 10 μl which was, in this case, half the volume of the reactionchamber. The anti-hCG and the goat anti-mouse latexes and gold conjugatewere added to the device and dried as described in Example 5. Clearplastic sheets of polycarbonate having a gap in one surface were placedon the devices with the gap facing the diagnostic element. Sample (about60 μl) containing 0 and 200 mIU hCG/ml was added to the devices. Thesample moved into the reaction chamber, dissolved the colloidal goldconjugate and the reaction mixture then moved onto the diagnosticelement over the anti-hCG latex. The reaction mixture then entered thegap which was immediately behind the capture zone of anti-hCG latex. Theflow rate over the capture zone slowed while the reaction mixture movedover the capture zone and filled the gap. The time for the 10 μlreaction mixture to fill the gap was about 12 min to 16 min, whereaswith devices without the optional flow control means, the times wereabout 1 min to 3 min. for the reaction mixture to pass over the capturezone. When the reaction mixture filled the gap, the reaction mixturethen moved into the narrow capillary of the diagnostic element and overthe goat anti-mouse capture zone. The reaction mixture moved into theused reagent reservoir and the excess sample washed the diagnosticelement. The color density of the capture zones for hCG was measuredinstrumentally using a Minolta Chroma Meter CR 241. A red color wasvisible for samples containing hCG and not visible for the samplewithout hCG at the capture zones for hCG. The ΔE* values for the 0 and200 mIU/ml were 0 and 16.12. The δE* value of the hCG capture zone forthe device without the flow control means for the 200 mIU/ml sample was16.32. A red color bar was visible at the goat anti-mouse IgG capturezones of each device.

EXAMPLE 8 Preparation of the Diagnostic Element for Multi-Step Assays

A device was built comprising a sample addition reservoir and adiagnostic element. The devices were plasma treated to graft functionalgroups which create a hydrophilic surface. The sample addition reservoirhad dimensions of 12 mm long, 6 mm wide and 0.05 mm deep. The diagnosticelement was about 5.5 cm long, 1.3 mm wide and 1 mm from the base of thedevice and included grooves running perpendicular to the flow ofreaction mixture at a density of 100 grooves per cm and a depth of 0.05mm. In the case of qualitative assays, the antibody latex (1 μl) wasapplied to the diagnostic element, covering the entire width and 1 cmlength of the diagnostic element. In the case of animmunochromatographic assay, the antibody latex (6 μl) was applied tothe entire width and length of the diagnostic element. The devices wereplaced under vacuum for about 1 h to dry the reagents. The capillaryspaces in the device were then formed by placing a clear plasticpolystyrene sheet on top of the device. The plastic sheet was held tothe opposing surface with binder clips.

EXAMPLE 9 Assay for hCG Using the Diagnostic Element

The diagnostic element described in Example 8 was used for the assay ofhCG. Urine samples (20 μl) containing 0, 50, 200 and 500 mIU/ml hCG wereadded to tubes containing anti-βhCG antibody colloidal gold conjugate (2μl). The tubes were vortexed and the reaction mixtures were incubatedfor 5 min at room temperature. The reaction mixtures (20 μl) wereapplied in 10 μl aliquots to the sample addition reservoir of thedevice. The reaction mixture flowed onto the diagnostic element from thesample reservoir and over the capture zone. An absorbent at the end ofthe capture zone removed the used reagent from the diagnostic element.The color density of the capture zones for hCG was measuredinstrumentally using a Minolta Chroma Meter CR 241. A red color wasvisible for samples containing hCG and not visible for the samplewithout hCG at the capture zones for hCG. The ΔE* values for the 0, 50,250 and 500 mIU/ml were 0.00, 1.24, 3.16 and 5.56, respectively.

EXAMPLE 10 Synthesis of meta-Nitrophencyclidine

To an ice cooled solution of phencyclidine hydrochloride (5 g, 1.8×10⁻²mol) in concentrated sulfuric acid (9 ml) was added dropwise, and withstirring, fuming nitric acid (2 ml). The reaction mixture was stirred inan ice-water bath for 1 hour and then poured onto crushed ice/water. Themixture was made basic with 10N sodium hydroxide (50 ml) to pH12 andextracted with diethyl ether (2×100 ml). The combined organic layerswere washed with water (2×100 ml), dried over anhydrous magnesiumsulfate, filtered and evaporated under vacuum. The residue was treatedwith methyl alcohol (20 ml) and heated on a hot water bath (80° C.)until solute dissolved. The flask was covered with aluminum foil(product is light sensitive) and the solution was allowed to stir atroom temperature overnight when a yellow solid precipitated. The solidwas collected by filtration and dried under vacuum to afford 3.0 g (58%)of m-nitrophencyclidine as fine yellow crystals which were protectedfrom light: mp 81-82° C.

EXAMPLE 11 Synthesis of meta-Aminophencyclidine

To a stirring solution of m-nitrophencyclidine (3.0 g, 10.4×10⁻³ mol) inmethyl alcohol (150 ml) was added, under a flow of argon, 10%palladium-carbon (0.5 g) followed by ammonium formate (4.0 g, 6.3×10⁻²mol). The reaction mixture was stirred at room temperature for 2 hoursafter which time the catalyst was removed by filtration and the solventwas evaporated under vacuum. The residue was treated with 1N potassiumhydroxide solution (30 ml) and extracted with diethyl ether (2×50 ml).The combined organic extracts were washed with water (50 ml), dried overanhydrous magnesium sulfate, filtered and evaporated under vacuum. Theresidue was dissolved in hexane (20 ml) and the solution was stirred atroom temperature overnight when a white solid precipitated. The solidwas collected by filtration and dried under vacuum to afford 1.4 g (52%)of m-aminophencyclidine: mp 121-122° C.

EXAMPLE 12 Synthesis of Acetylthiopropionic Acid

To a stirred solution of 3-mercaptoproprionic acid (7 ml, 0.08 moles)and imidazole (5.4 g, 0.08 moles) in tetrahydrofuran (THF, 700 ml) wasadded dropwise over 15 minutes, under argon, a solution of 1-acetylimidazole (9.6 g, 0.087 moles) in THF (100 ml). The solution was allowedto stir a further 3 hours at room temperature after which time the THFwas removed in vacuo. The residue was treated with ice-cold water (18ml) and the resulting solution acidified with ice-cold concentrated HCl(14.5 ml) to pH 1.5-2. The mixture was extracted with water (2×50 ml),dried over magnesium sulfate and evaporated. The residual crude yellowoily solid product (10.5 g) was recrystallized from chloroform-hexane toafford 4.8 g (41% yield) acetylthiopropionic acid as a white solid witha melting point of 44-45° C.

EXAMPLE 13 Synthesis of meta-Acetylthiopropionamide Phencyclidine

To a stirring solution of m-aminophencyclidine (1.4 g, 5.4×10⁻³ mol) andacetylthiopropionic acid (0.87 g, 5.8×10⁻³ mol) in anhydroustetrahydrofuran (7 ml) was added dicyclohexylcarbodiimide (1.19 g,5.8×10⁻³ mol). The flask was purged with argon and the solution stirredat room temperature for 2 hours. The mixture was filtered from insolubledicyclohexylurea and evaporated under vacuum. The residual solid wasrecrystallized from chloroform/hexane to afford 1.5 g (71%) ofm-acetylthiopropionamide phencyclidine as a white crystalline solid:mp152-4° C.

EXAMPLE 14 Synthesis of meta-3-Mercaptoproprionamide Phencyclidine

meta-Acetylthiopropionamide phencyclidine (0.01 g, 2.57×10⁻⁵ mol) wasdissolved in 1.29 ml 0.12M potassium carbonate in 80% methanol/20% water(v/v). The solution sat at room temperature for 5 min and then 0.2 ml0.5 M potassium phosphate, pH 7, was immediately added and the solutionwas adjusted to pH 7-7.5 with hydrochloric acid (1 N). The titlecompound in solution was used as is to react with BSA-SMCC.

EXAMPLE 15 Preparation of Phencyclidine Analogue Attached to BovineSerum Albumin (BSA-PCP)

Bovine serum albumin (BSA, 3.5 ml of 20 mg/ml) was reacted withsuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC,Pierce Chemical Co.) by adding a solution of 6.7 mg SMCC in 0.3 mlacetonitrile and stirring the solution at room temperature for 1 h whilemaintaining the pH between 7 and 7.5 with 1N potassium hydroxide. Theprotein was separated form unreacted compounds by gel filtrationchromatography in 0.1 M potassium phosphate, 0.02 M potassium borate,0.15 M sodium chloride, pH 7.0. The meta-3-mercaptoproprionamidephencyclidine (0.2 ml of 13 mM) was added to the BSA-maleimide (2 ml at8.2 mg/ml) and the solution was stirred at room temperature for 4 h. Thesolution was then dialyzed 3 times against 1000 ml of 10 mM MES, pH 5.5.Recover 1.8 ml BSA-PCP at 8 mg/ml.

EXAMPLE 16 Preparation of Phencyclidine Analogue Colloidal GoldConjugate

A solution (4.7 ml) containing BSA (22 mg) and BSA-PCP (5.6 mg) in 10 mMMES, pH 5.5 was added in a bolus to colloidal gold (105 ml) in 10 mMMES, pH 5.5 with rapid stirring. After complete mixing the stirring wasstopped and the solution was incubated at room temperature for 1 h. Thecolloidal gold conjugate was subjected to diafiltration against 50 mMpotassium phosphate, 10 mM potassium borate, pH 7, using a tangentialflow device (Sartorius Easy Flow, molecular weight cutoff was 100,000)to remove BSA and BSA-PCP which was not bound to colloidal gold. Thegold conjugate was diluted for the assay of PCP into a buffered solutioncontaining 10 mg/ml bovine serum albumin at pH 7.5.

EXAMPLE 17 Preparation of anti-Phencyclidine Antibody Latex

Surfactant-free polystyrene particles (Interfacial Dynamics Corp.,Portland, Oreg.; 0.074 ml of 9.4% solids, 0.4 μm) was added whilevortexing to anti-phencyclidine monoclonal antibody (0.926 ml of 5.86mg/ml in 0.1 M MES, pH 5) and the suspension was incubated at 45° C. for2 h. The suspension was subjected to centrifugation to pellet the latexparticles. The pellet was washed three times by centrifugation andresuspension of the pellet with 10 mM MES, 0.1 mg/ml trehalose, pH 5.5.The final pellet was resuspended in the wash buffer at a solidsconcentration of 1%.

EXAMPLE 18 Preparation of Latex-Immobilized Affinity-Purified Goat IgGAntibody Against the Fc Fragment of Mouse IgG (Goat Anti-Mouse Fc Latex)

Affinity-purified goat anti-mouse (Fc (Immunosearch) and polystyrenelatex particles (sulfated, 1.07 μm) (Interfacial Dynamics) wereincubated separately at 45° C. for one hour, the antibody solution beingbuffered with 0.1 M 2-(N-morpholino) ethane sulfonic acid at pH 5.5.While vortexing the antibody solution, the solution of latex particleswas added to the antibody solution such that the final concentration ofantibody was 0.3 mg/ml and the solution contained 1% latex solids. Thesuspension was incubated for 2 hours at 45° C. prior to centrifugationof the suspension to pellet the latex particles. The latex pellet wasresuspended in 1% bovine serum albumin in phosphate-buffered-saline(PBS) and incubated for one hour at room temperature. Followingcentrifugation to pellet the latex, the pellet was washed three times byresuspension in PBS and centrifugation. The final pellet was resuspendedin PBS containing 0.1% sodium azide at pH 7.0 at a latex concentrationof 1% solids.

EXAMPLE 19 Assay for Phencyclidine Using the Diagnostic Element

The diagnostic element described in Example 8 was used for the assay ofphencyclidine (PCP). Urine samples (133 μl) containing 0, 100, 200 and300 ng/ml PCP were added to tubes containing a lyophilized bufferformulation (containing 10 mM potassium phosphate, 150 mM sodiumchloride and 10 mg/ml BSA, pH 8) and phencyclidine analogue colloidalgold conjugate (4 μl) was added and the solution was vortexed. Anti-PCPantibody (2.8 μl of 0.1 mg/ml) was added to each tube and the solutionswere vortexed and incubated at room temperature for 5 min. Goatanti-mouse Fc latex (50 ml of a 1% suspension) was added to the tubes,the tubes were vortexed and incubated at room temperature for 10 min.The solutions were then filtered to remove the complex of the PCPanalogue gold conjugate: anti-PCP antibody:goat anti-mouse latex fromthe reaction mixture using a Gelman Acrodisc® 3 syringe filter (0.45μm). The filtrates of the reaction mixtures (20 μl) were applied to thediagnostic elements described in example 8. The reaction mixture flowedonto the diagnostic element from the sample reservoir and over thecapture zone. An absorbent tissue placed 1 cm after the capture zoneremoved the used reagent from the diagnostic element. The color densityof the capture zones was measured instrumentally using a Minolta ChromaMeter CR 241. The ΔE* values for the 0, 100, 200 and 300 ng/ml sampleswere 0.69, 9.28, 14.04 and 21.6, respectively.

Although the foregoing invention has been described in some detail byway of illustration and example, it will be obvious that certain changesor modifications may be practiced within the scope of the appendedclaims. As used herein, references to "preferred" embodiments refer tobest modes for practicing the invention.

What is claimed is:
 1. A diagnostic assay device for detecting a targetligand in a reaction mixture, said device comprising:i. a sampleaddition reservoir; ii. a non-porous diagnostic element located within acapillary space, through which all of said reaction mixture flows,wherein said reaction mixture flows from said sample addition reservoirto said diagnostic element without application of an external force, andwherein said diagnostic element immobilizes for detection at least oneconjugate in an amount related to the amount of target ligand in thereaction mixture in at least one zone.
 2. A test device comprising:i) asample addition reservoir comprising a capillary space; ii) a diagnosticelement comprising two non-porous surfaces that form a capillary of from1 micron to 100 microns and having at least one zone comprised of abiological material which reacts with a target ligand or a conjugate ofthe target ligand; and iii) a used reagent reservoir, wherein when areaction mixture is added to said sample addition reservoir, saidreaction mixture flows through said diagnostic element to said usedreagent reservoir such that substantially all of said reaction mixturepasses over said at least one zone in said diagnostic element and anamount of the target ligand present in said reaction mixture related tothe presence or amount of the target ligand in said reaction mixture iscaptured for detection in said at least one zone in said diagnosticelement.
 3. A diagnostic assay device comprising:a diagnostic elementhaving a nonabsorbent surface for detection of at least one targetligand in at least one zone, the nonabsorbent surface having particlesimmobilized thereon, which particles comprise receptor that reacts withsaid at least one target ligand, wherein when a reaction mixturecomprising said at least one target ligand is added to the device, saidreaction mixture contacts said immobilized particles, and an amount ofsaid at least one target ligand is captured for detection by saidreceptor.
 4. The device of claim 3 wherein the particles are latex. 5.The device of claim 3 wherein the particles are polystyrene.
 6. Thedevice of claim 3 wherein the particles are nanoparticles.
 7. The deviceof claim 6 wherein the nanoparticles comprise silica, zirconia, alumina,titania, ceria, metal sols, or polystyrene.
 8. The device of claim 6wherein the nanoparticles have sizes in a range from about 1 nm to 100nm.
 9. The device of claim 6 wherein the nanoparticles are immobilizedon said nonabsorbent surface through adsorption or covalent bonds. 10.The device of claim 3 wherein said particles are immobilized on saidnonabsorbent surface by magnetic means, hydrophobic means, hydrogenbonding, electrostatic means, or entrapment.
 11. The device of claim 3,wherein said particles have diameters ranging from about 0.1 mm to 10mm.
 12. The device of claim 3, wherein said device comprises the form ofa dipstick.
 13. The device of claim 3, wherein said receptor isimmobilized on a surface of the particle.
 14. The device of claim 13,wherein said surface binds a proteinaceous component.
 15. The device ofclaim 14, wherein said surface binds gelatin, serum albumins,immunoglobulins, enzymes, polypeptides, and hydrophilic polymers. 16.The device of claim 15, wherein said surface binds bovine serum albumin.17. The device of claim 3, wherein said receptor comprises an antibody.18. The device of claim 17, wherein said receptor comprises a monoclonalantibody.
 19. The device of claim 17, wherein said receptor comprises agoat Immunoglobulin G antibody that binds mouse Immunoglobulin G.
 20. Adevice of claim 18, wherein said receptor comprises an anti humanchorionic gonadotropin α-subunit monoclonal antibody.
 21. The device ofclaim 18, wherein said receptor comprises an anti-phencyclidinemonoclonal antibody.